The invention relates to novel cytidine deaminase-like nucleic acid sequences and proteins. Also provided are vectors, host cells, and recombinant methods for making and using the novel molecules.
Cytidine deaminases convert, by irreversible hydrolytic deamination, cytidine and deoxycytidine to uridine and deoxyuridine, respectively. The deamination of cytidine has been demonstrated to affect numerous mammalian cellular processes. For instance, increasing evidence suggests that base-modification of mRNA editing occurs through deamination via cytidine deaminases. Current data suggests that unregulated editing or inappropriate occurrence of mRNA editing can lead to various disease states. For a review see, for example, Smith et al. (1996) Trends in Genetics 12:418-424. Among vertebrates, examples of mRNA editing resulting in a cytidine to uridine conversion include APOB mRNA in rat liver and intestine (Chen et al. (1987) Science 328:363-366 and Powell et al. (1987) Cell 50:831-840); Neurofibromatosis type-1 mRNA in human tumors (Skuse et al. (1996) Nucleic Acid Res. 24:478-486); tRNAASP in Rat liver (Beier et al. (1992) Nucleic Acid Research 20:2679-2683); and tRNAGLY in marsupials (Janke et al. (1993) Nucleic Acid 21:1523-1525).
The mRNA editing process of the APOB mRNA has been well characterized. The base modification of APOB mRNA occurs through hydrolytic removal of the C4 and C6 amino groups from cytidine. The editing event results in two isoforms of the protein, APOB100 and APOB48. The APOB protein binds lipid to form lipoprotein particles and, thereby, participates in the transports of lipids to peripheral body tissues. Lipoprotein particles assembled on APOB48 are cleared from the blood more rapidly than those containing APOB100 and are not metabolized to the low density lipoprotein (LDL). The cytidine deamination event of the APOB mRNA therefore influences the level of LDL in the blood stream and may ultimately influence the development of atherogenic diseases (Davison et al. (1993) Ann. Med. 25:539-543 and Greeve et al (1993) J. Lipid Res. 34:1367-1383).
Biochemical analysis of the APOB mRNA editing process has demonstrate that the cytidine deaminase, APOBEC1 (APOB-editing catalytic subunit 1), plays a critical role in the deamination event. Current evidence suggests that APOBEC1 alone can only deaminate cytidine mononucleotides (Navaratnam et al. (1993) J. Biol. Chem. 268:20709-20712). It appears that the ability to edit APOB mRNA is imposed upon APOCEC1 allosterically through its assembly with other protein (auxiliary factors) as holoenzymes editing complexes or xe2x80x9ceditosomesxe2x80x9d.
Other cytidine deamination mRNA editing events have been implicated in cell cycle regulation. The editing site of NF1 mRNA is 67% identical to that found in APOB mRNA. A cytidine to uridine conversion in the NF1 mRNA results in a truncated translation product. The truncated product is unable to maintain RAS in the inactive GDP form, and would therefore, presumably, be unable to regulate cell proliferation (Skuse et al. (1996) Nucleic Acid Research 24:478-486).
In addition to its natural substrates, cytidine and deoxycytidine, cytidine deaminases also catalyzes the deamination of cytosine nucleoside analogs including the antineoplastic agents ARA-C, dFdC, and 5-AZA-CdR. The deamination of these compounds results in a loss of their pharmacological activity. Cytidine deaminases may therefore have the ability to confer resistance to these drugs and potentially circumvent the hematopoietic toxicity produced by chemotherapy with cytosine nucleoside analogs.
Recently, in vitro data has demonstrated that it is possible to confer drug resistance to cytosine nucleoside analogs by the transfer of the cytidine deaminase gene into murine hematopoietic cells. Further in vivo studies have demonstrated that is possible to transplant cytidine deaminase-transduced marrow cells into mice and obtain long-term increased expression of cytidine deaminase in hematopoietic cells. For a review see Bertion et al. (1999) Prog Exp Tum Res 36:124-142. Therefore, cytidine deaminases have potential to render normal hematopoietic cells resistant to cytosine nucleoside analogs and thereby increase the clinical efficacy of these chemotherapeutic drugs in cancer treatment.
Cytidine deaminase family members play critical regulatory roles in various cellular processes. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize novel cytidine deaminases. The present invention advances the state of the art by providing a novel human cytidine deaminase-like nucleic acid and polypeptide.
Isolated nucleic acid molecules corresponding to cytidine deaminase-like nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO:2. Further provided are cytidine deaminase-like polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein.
The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as methods of making such vectors and host cells and for using them for production of the polypeptides or peptides of the invention by recombinant techniques.
The cytidine deaminase-like molecules of the present invention are useful as modulating agents in a variety of cellular processes including cytidine deamination and mRNA editing. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding cytidine deaminase-like proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of cytidine deaminase-like-encoding nucleic acids.
Another aspect of this invention features isolated or recombinant cytidine deaminase-like proteins and polypeptides. Preferred cytidine deaminase-like proteins and polypeptides possess at least one biological activity possessed by naturally occurring cytidine deaminase-like proteins.
Variant nucleic acid molecules and polypeptides substantially homologous to the nucleotide and amino acid sequences set forth in the sequence listings are encompassed by the present invention. Additionally, fragments and substantially homologous fragments of the nucleotide and amino acid sequences are provided.
Antibodies and antibody fragments that selectively bind the cytidine deaminase-like polypeptides and fragments are provided. Such antibodies are useful in detecting the cytidine deaminase-like polypeptides as well as in regulating cytidine deamination events, such as those that occur during mRNA editing.
In another aspect, the present invention provides a method for detecting the presence of cytidine deaminase-like activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of cytidine deaminase-like activity such that the presence of cytidine deaminase-like activity is detected in the biological sample.
In yet another aspect, the invention provides a method for modulating cytidine deaminase-like activity comprising contacting a cell with an agent that modulates (inhibits or stimulates) cytidine deaminase-like activity or expression such that cytidine deaminase-like activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to cytidine deaminase-like protein. In another embodiment, the agent modulates expression of cytidine deaminase-like protein by modulating transcription of an cytidine deaminase-like gene, splicing of an cytidine deaminase-like mRNA, or translation of an cytidine deaminase-like mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the cytidine deaminase-like mRNA or the cytidine deaminase-like gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant cytidine deaminase-like protein activity or nucleic acid expression by administering an agent that is an cytidine deaminase-like modulator to the subject. In one embodiment, the cytidine deaminase-like modulator is an cytidine deaminase-like protein. In another embodiment, the cytidine deaminase-like modulator is an cytidine deaminase-like nucleic acid molecule. In other embodiments, the cytidine deaminase-like modulator is a peptide, peptidomimetic, or other small molecule.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of the following: (1) aberrant modification or mutation of a gene encoding an cytidine deaminase-like protein; (2) misregulation of a gene encoding an cytidine deaminase-like protein; and (3) aberrant post-translational modification of an cytidine deaminase-like protein, wherein a wild-type form of the gene encodes a protein with an cytidine deaminase-like activity.
In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of an cytidine deaminase-like protein. In general, such methods entail measuring a biological activity of an cytidine deaminase-like protein in the presence and absence of a test compound and identifying those compounds that alter the activity of the cytidine deaminase-like protein.
The invention also features methods for identifying a compound that modulates the expression of cytidine deaminase-like genes by measuring the expression of the cytidine deaminase-like sequences in the presence and absence of the compound.
Other features and advantages of the invention will be apparent from the following detailed description and claims.